A precise understanding of the sequence-stability relationship is of fundamental interest in protein biochemistry, as protein instability is a cause f a wide range of pathologies, and it would enable facile engineering of proteins for industrial and therapeutic purposes. Protein engineering and de novo design have broadly delineated the forces that stabilize proteins and have yielded some spectacular successes in designing new or stabilized proteins. However, we are still far from a precise physicochemical model of protein stability; there is still no reliable way to predict the thermodynamic consequences of an arbitrary mutation. In the first five years of this project, we established a novel high-throughput dye binding method (HTTS) to measure protein stability, and applied it and a cell-based screen for function to the study of core, loop and surface variants of the well-studied homodimeric four-helix bundle protein Rop. This strategy enabled us to directly test hypotheses of protein design, by building large, targeted libraries of protein variants and sorting them for foldedness. We designed a Cys-free Rop as the scaffold for this work, which was used in Rop topology studies, and we designed an active, stable single-chain Rop. We developed a bacterial screen for the DNA binding activity of the core domain of the tumor suppressor p53, and used it to screen a small core library. Here we propose to extend studies of Rop by drilling down into surprising discoveries about the stability effects of disturbances of the heptad repeat and the prevalence of Val residues in the core; a stabilizing i to i+2 electrostatic interaction in the loop; and the prevalence of Lys in selected variants in surface positions. These studies will be informed by collaborations in computational protein design. We also will explore more subtle effects at different positions in the heptad repeat, and repacking around low-propensity and unnatural amino acids. We are developing and innovative all-in vitro version of HTTS that couples gene synthesis, in vitro transcription/translation directly to HT purification and stability measurement giving us unprecedented control over our libraries. These studies can be combined with our Rop enrichment selection to directly compare fitness and stability with deep sequencing and HTTS. We will use the p53 screen for similar studies, especially focusing on the role of a loop in dynamics and stability. Finally, we are developing HT screening systems for Cra and Rosetta-designed proteins to interface our studies with statistical and computational protein design. The throughput of biophysical characterization is poor, so only a small number of protein mutants have been examined in detail for most scaffolds. This prevents thorough study of effects such as sequence correlation or exploration of shallower energy surfaces. Here, we will continue to use the power of high- throughput approaches to test and refine protein design principles with statistical significance, both improving our knowledge of the sequence-structure relationship and enabling future design and therapeutic approaches. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page